Ipsilateral Spinal Dorsal Horn Research Articles

BMP10 accelerated spinal astrocytic activation in neuropathic pain via ALK2/smad1/5/8 signaling.

Astrocytic activation in the spinal dorsal horn contributes to the central sensitization of neuropathic pain. Bone morphogenetic protein (BMP) 10, one of the BMPs highly expressed in the central nervous system, has been demonstrated to have an accelerated effect on astrocytic activation. This study aimed to investigate the functional effects of BMP10 on the activation of astrocytes in the spinal dorsal horn of animal model of neuropathic pain and to explore potential mechanisms involved in this process. A neuropathic pain mice model was established using the spared nerve injury (SNI). Western blot analysis was performed to detect the expressional levels of BMP10, activin receptor-like receptor 2 (ALK2), Smad1/5/8, phosphorylated Smad1/5/8, and glial fibrillary acidic protein (GFAP). Immunofluorescence staining was used to detect BMP10, ALK2, and GFAP distribution and expression. The behavioral changes in mice were evaluated using paw withdrawal threshold (PWT), thermal withdrawal latency (TWL), and open field test (OFT). The BMP10 siRNA, Smad1 siRNA, BMP10 peptide, and ALK2-IN-2 (ALK2 inhibitor) were intrathecally administrated to mice. A model of lipopolysaccharide (LPS)-stimulated astrocytes was established to investigate the effect of Smad1. The transfection efficiency of siRNAs was detected by western blot and qRT-PCR analysis. BMP10 levels were increased in the L4-6 ipsilateral spinal dorsal horn of SNI mice and particularly elevated in astrocytes. Consistently, GFAP and phosphorylated Smad1/5/8 were upregulated in the L4-6 ipsilateral spinal dorsal horn after SNI, indicating the activation of astrocytes and Smad1/5/8 signaling. An intrathecal injection of BMP10 siRNA abrogated pain hypersensitivity and astrocytic activation in SNI mice. In addition, intrathecal administration of BMP10 peptide evoked pain hypersensitivity and astrocytic activation in normal mice, and this action was reversed by inhibiting the ALK2. Furthermore, targeting Smad1 in vitro with the help of siRNA inhibited the activation of astrocytes induced by LPS. Finally, targeting Smad1 abrogated BMP10-induced hypersensitivity and activation of astrocytes. These findings indicate that the BMP10/ALK2/Smad1/5/8 axis plays a key role in pain hypersensitivity after peripheral nerve injury, which indicates its stimulative ability toward astrocytes.

Intrathecal Fumagillin Alleviates Chronic Neuropathy-Induced Nociceptive Sensitization and Modulates Spinal Astrocyte-Neuronal Glycolytic and Angiogenic Proteins.

Nociceptive sensitization is accompanied by the upregulation of glycolysis in the central nervous system in neuropathic pain. Growing evidence has demonstrated glycolysis and angiogenesis to be related to the inflammatory processes. This study investigated whether fumagillin inhibits neuropathic pain by regulating glycolysis and angiogenesis. Fumagillin was administered through an intrathecal catheter implanted in rats with chronic constriction injury (CCI) of the sciatic nerve. Nociceptive, behavioral, and immunohistochemical analyses were performed to evaluate the effects of the inhibition of spinal glycolysis-related enzymes and angiogenic factors on CCI-induced neuropathic pain. Fumagillin reduced CCI-induced thermal hyperalgesia and mechanical allodynia from postoperative days (POD) 7 to 14. The expression of angiogenic factors, vascular endothelial growth factor (VEGF) and angiopoietin 2 (ANG2), increased in the ipsilateral lumbar spinal cord dorsal horn (SCDH) following CCI. The glycolysis-related enzymes, pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) significantly increased in the ipsilateral lumbar SCDH following CCI on POD 7 and 14 compared to those in the control rats. Double immunofluorescence staining indicated that VEGF and PKM2 were predominantly expressed in the astrocytes, whereas ANG2 and LDHA were predominantly expressed in the neurons. Intrathecal infusion of fumagillin significantly reduced the expression of angiogenic factors and glycolytic enzymes upregulated by CCI. The expression of hypoxia-inducible factor-1α (HIF-1α), a crucial transcription factor that regulates angiogenesis and glycolysis, was also upregulated after CCI and inhibited by fumagillin. We concluded that intrathecal fumagillin may reduce the expression of ANG2 and LDHA in neurons and VEGF and PKM2 in the astrocytes of the SCDH, further attenuating spinal angiogenesis in neuropathy-induced nociceptive sensitization. Hence, fumagillin may play a role in the inhibition of peripheral neuropathy-induced neuropathic pain by modulating glycolysis and angiogenesis.

Osteosarcoma-Induced Pain Is Mediated by Glial Cell Activation in the Spinal Dorsal Horn, but Not Capsaicin-Sensitive Nociceptive Neurons: A Complex Functional and Morphological Characterization in Mice.

Bone cancer and its related chronic pain are huge clinical problems since the available drugs are often ineffective or cannot be used long term due to a broad range of side effects. The mechanisms, mediators and targets need to be identified to determine potential novel therapies. Here, we characterize a mouse bone cancer model induced by intratibial injection of K7M2 osteosarcoma cells using an integrative approach and investigate the role of capsaicin-sensitive peptidergic sensory nerves. The mechanical pain threshold was assessed by dynamic plantar aesthesiometry, limb loading by dynamic weight bearing, spontaneous pain-related behaviors via observation, knee diameter with a digital caliper, and structural changes by micro-CT and glia cell activation by immunohistochemistry in BALB/c mice of both sexes. Capsaicin-sensitive peptidergic sensory neurons were defunctionalized by systemic pretreatment with a high dose of the transient receptor potential vanilloid 1 (TRPV1) agonist resiniferatoxin (RTX). During the 14- and 28-day experiments, weight bearing on the affected limb and the paw mechanonociceptive thresholds significantly decreased, demonstrating secondary mechanical hyperalgesia. Signs of spontaneous pain and osteoplastic bone remodeling were detected both in male and female mice without any sex differences. Microglia activation was shown by the increased ionized calcium-binding adapter molecule 1 (Iba1) immunopositivity on day 14 and astrocyte activation by the enhanced glial fibrillary acidic protein (GFAP)-positive cell density on day 28 in the ipsilateral spinal dorsal horn. Interestingly, defunctionalization of the capsaicin-sensitive afferents representing approximately 2/3 of the nociceptive fibers did not alter any functional parameters. Here, we provide the first complex functional and morphological characterization of the K7M2 mouse osteosarcoma model. Bone-cancer-related chronic pain and hyperalgesia are likely to be mediated by central sensitization involving neuroinflammation via glial cell activation in the spinal dorsal horn, but not the capsaicin-sensitive sensory neuronal system.

Local Magnesium Sulfate Administration Ameliorates Nociception, Peripheral Inflammation, and Spinal Sensitization in a Rat Model of Incisional Pain

ObjectivePostoperative pain remains one of the most common complaints after surgery, and appropriate treatments are limited. MethodsWe therefore investigated the effect of the anti-nociceptive properties of magnesium sulfate (MgSO4), an N-methyl-D-aspartate (NMDA) receptor antagonist, on incision-induced postoperative pain and peripheral and central nervous system inflammation. ResultsWe found that local MgSO4 administration dose-dependently increases paw withdrawal latency, indicating reduced peripheral postoperative pain. Furthermore, MgSO4 inhibited the expression of interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) and phosphorylation of the NMDA receptor NR1 subunit in injured paw tissue and significantly attenuated microglial and astrocytic activation in the ipsilateral lumbar spinal cord dorsal horn. ConclusionLocally administered MgSO4 has potential for development as an adjunctive therapy for preventing central nociceptive sensitization.

PRMT6 deficiency or inhibition alleviates neuropathic pain by decreasing glycolysis and inflammation in microglia

Microglia induced chronic inflammation is the critical pathology of Neuropathic pain (NP). Metabolic reprogramming of macrophage has been intensively reported in various chronic inflammation diseases. However, the metabolic reprogramming of microglia in chronic pain remains to be elusive. Here, we reported that immuno-metabolic markers (HIF-1α, PKM2, GLUT1 and lactate) were related with increased expression of PRMT6 in the ipsilateral spinal cord dorsal horn of the chronic construction injury (CCI) mice. PRMT6 deficiency or prophylactic and therapeutic intrathecal administration of PRMT6 inhibitor (EPZ020411) ameliorated CCI-induced NP, inflammation and glycolysis in the ipsilateral spinal cord dorsal horn. PRMT6 knockout or knockdown inhibited LPS-induced inflammation, proliferation and glycolysis in microglia cells. While PRMT6 overexpression exacerbated LPS-induced inflammation, proliferation and glycolysis in BV2 cells. Recent research revealed that PRMT6 could interact with and methylate HIF-1α, which increased HIF-1α protein stability. In sum, increased expression of PRMT6 exacerbates NP progress by increasing glycolysis and neuroinflammation through interacting with and stabilizing HIF-1α in a methyltransferase manner, which outlines novel pathological mechanism and drug target for NP.

Massage ameliorates lumbar disc herniation-related radicular pain in rats by suppressing TLR4/NLRP3 inflammasome signaling transduction.

Lumbar disc herniation (LDH) is a common spinal disease that can cause severe radicular pain. Massage, also known as Tuina in Chinese, has been indicated to exert an analgesic effect in patients with LDH. Nonetheless, the mechanism underlying this effect of massage on LDH remains unclarified. Forty Sprague-Dawley rats were randomly divided into four groups. A rat LDH model was established by autologous nucleus pulpous (NP) implantation, followed by treatment with or without massage. A toll-like receptor 4 (TLR4) antagonist TAK-242 was administrated to rats for blocking TLR4. Behavioral tests were conducted to examine rat mechanical and thermal sensitivities. Western blotting was employed for determining TLR4 and NLRP3 inflammasome-associated protein levels in the spinal dorsal horn (SDH). Immunofluorescence staining was implemented for estimating the microglial marker Iba-1 expression in rat SDH tissue. NP implantation induced mechanical allodynia and thermal hyperalgesia in rat ipsilateral hindpaws and activated TLR4/NLRP3 inflammasome signaling transduction in the ipsilateral SDH. Massage therapy or TAK-242 administration relieved NP implantation-triggered pain behaviors in rats. Massage or TAK-242 hindered microglia activation and blocked TLR4/NLRP3 inflammasome activation in ipsilateral SDH of LDH rats. Massage ameliorates LDH-related radicular pain in rats by suppressing microglia activation and TLR4/NLRP3 inflammasome signaling transduction.

HK2 in microglia and macrophages contribute to the development of neuropathic pain.

Neuropathic pain is a complex pain condition accompanied by prominent neuroinflammation involving activation of both central and peripheral immune cells. Metabolic switch to glycolysis is an important feature of activated immune cells. Hexokinase 2 (HK2), a key glycolytic enzyme enriched in microglia, has recently been shown important in regulating microglial functions. Whether and how HK2 is involved in neuropathic pain-related neuroinflammation remains unknown. Using a HK2-tdTomato reporter line, we found that HK2 was prominently elevated in spinal microglia. Pharmacological inhibition of HK2 effectively alleviated nerve injury-induced acute mechanical pain. However, selective ablation of Hk2 in microglia reduced microgliosis in the spinal dorsal horn (SDH) with little analgesic effects. Further analyses showed that nerve injury also significantly induced HK2 expression in dorsal root ganglion (DRG) macrophages. Deletion of Hk2 in myeloid cells, including both DRG macrophages and spinal microglia, led to the alleviation of mechanical pain during the first week after injury, along with attenuated microgliosis in the ipsilateral SDH, macrophage proliferation in DRGs, and suppressed inflammatory responses in DRGs. These data suggest that HK2 plays an important role in regulating neuropathic pain-related immune cell responses at acute phase and that HK2 contributes to neuropathic pain onset primarily through peripheral monocytes and DRG macrophages rather than spinal microglia.

Comparisons of the analgesic effect of different pulsed radiofrequency targets in SNI-induced neuropathic pain.

An injury of the peripheral nerve may lead to neuropathic pain, which could be treated with pulsed radiofrequency to the dorsal root ganglion (DRG) or peripheral nerve [the nerve trunk (NT) or proximal to the injury site (NI)]. However, it is not clear whether there is any difference in analgesic effect or maintenance among the three targets. PRF was applied to the ipsilateral L5 DRG, peripheral nerve (NT or NI) 5 days after spared nerve injury (SNI). Triptolide (10 µg/kg) or vehicle was intrathecally administered 5 days after SNI for 3 days. Mechanical withdrawal thresholds were tested after treatment at different time points. Furthermore, microglia and the P2X7 receptor (P2X7R) in the ipsilateral spinal cord were measured with immunofluorescence and western blotting, respectively. PRF + NI exerted a more remarkable analgesic effect than PRF + DRG and PRF + NT at the early stage, but PRF + DRG had a stronger analgesic effect than PRF + NI and PRF + NT at the end of our study. In addition, PRF + DRG showed no significant difference from intrathecal administration of triptolide. Moreover, SNI-induced microglia activation and upregulation of P2X7R in spinal dorsal horn could be markedly inhibited by PRF + DRG. The results suggest that the analgesic effect of PRF + DRG increased with time whereas the other two not and microglia and P2X7R in the ipsilateral spinal dorsal horn may be involved in the process.

Characterization of pain-related behaviors and gene expression profiling of peripheral sensory ganglia in a mouse model of acute ankle sprain.

Lateral ankle sprain (LAS) is a very common type of joint injury. It occurred with high incidence among general population and especially among individuals participating sports and outdoor activities. A certain proportion of individuals who once developed LAS may suffer persistent ankle pain that affects daily activities. However, the mechanisms underlying LAS-induced pain still remained largely unknown. We established a LAS mouse model and systematically evaluated the pain-related behaviors in this mouse model. RNA sequencing (RNA-Seq), combined with bioinformatics analysis, was undertaken to explore gene expression profiles. Immunostaining was used to study glial cell and neuron activation in ipsilateral spinal cord dorsal horn (SCDH) of LAS model mice. Ibuprofen was used to treat LAS model mice. The LAS model mice developed obvious signs of mechanical and heat hypersensitivities as well as gait impairments in ipsilateral hind paws. Besides, LAS model mice developed signs of pain-related emotional disorder, including pain-induced aversion. By RNA-Seq, we were able to identify certain differentially expressed genes and signaling pathways that might contribute to pain mechanisms of LAS mouse model. In addition, LAS model mice showed increased c-Fos and p-ERK immunoreactivity as well as astrocyte and microglia overactivation in ipsilateral spinal cord dorsal horn, indicating central sensitization might occur. Finally, LAS model mice respond to ibuprofen, a drug clinically used to treat ankle sprain pain. Our study found LAS model mice may be used as a preclinical animal model for screening novel targets or therapies for ankle sprain. Thus, the study may further help to understand molecular mechanisms contributing to ankle sprain-induced pain.

Folic acid relieves bone cancer pain by downregulating P2X2/3 receptors in rats

BackgroundBone cancer pain (BCP) remains a clinical challenge due to the limited and side effects of therapeutic methods. Folic acid has been known as an FDA approved dietary supplement and proved to have an analgesic effect in neuropathic pain. Here we investigate the role and mechanism of folic acid in bone cancer pain of a rat model. MethodsWalker 256 tumor cells were inoculated into the left tibia of rats to induce bone cancer pain model. Pain reflex were assessed by paw withdrawal threshold (PWT) response to Von Frey filaments and paw withdrawal latency (PWL) response to thermal stimulation. Folic acid was injected intraperitoneally to evaluate its analgesic effect in rats with bone cancer pain. Western blotting and qPCR were used to determine P2X2/3 receptor protein and mRNA levels in ipsilateral L4-6 dorsal root ganglion (DRG) and spinal dorsal horn (SDH). ResultsThe PWT and PWL of rats with bone cancer pain were obviously decreased compared to the naïve and sham rats. Interestingly, continuous folic acid treatment significantly increased the PWT and PWL of rats with bone cancer pain. P2X2 and P2X3 receptors were clearly upregulated at both mRNA and protein expression in L4-6 DRG and SDH of rats with bone cancer pain. P2X2 and P2X3 receptors were mainly localized with CGRP (calcitonin gene-related peptide) or IB4 (isolectin B4) positive neurons in L4-6 DRG of rats with bone cancer pain. Notably, continuous folic acid treatment significantly reduced the expression of P2X2 and P2X3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Finally, intrathecal injection of A317491 (a selective antagonist of P2X2/3 receptors) markedly elevated the PWT and PWL of rats with bone cancer pain. ConclusionThese results suggest that folic acid has an effective antinociceptive effect on bone cancer pain, which is mediated by downregulating P2X2/3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Folic acid may be a novel therapeutic strategy in cancer patients for pain relief.

Differential activation of spinal and parabrachial glial cells in a neuropathic pain model.

The clinical burden faced by chronic pain patients is compounded by affective comorbidities, such as depression and anxiety disorders. Emerging evidence suggests that reactive glial cells in the spinal cord dorsal horn play a key role in the chronification of pain, while supraspinal glia are important for psychological aspects of chronic pain. The lateral parabrachial nucleus (LPBN) in the brainstem is a key node in the ascending pain system, and is crucial for the emotional dimension of pain. Yet, whether astrocytes and microglia in the LPBN are activated during chronic pain is unknown. Here, we evaluated the occurrence of glial activation in the LPBN of male Sprague-Dawley rats 1, 4, and 7 weeks after inducing a chronic constriction injury (CCI) of the sciatic nerve, a prevalent neuropathic pain model. CCI animals developed mechanical and thermal hypersensitivity that persisted for at least 4 weeks, and was mostly reversed after 7 weeks. Using immunohistochemical staining and confocal imaging, we found that CCI caused a strong increase in the expression of the astrocytic marker GFAP and the microglial marker Iba1 in the ipsilateral spinal dorsal horn, with peak expression observed 1 week post-injury. Moreover, morphology analysis revealed changes in microglial phenotype, indicative of microglia activation. In contrast, CCI did not induce any detectable changes in either astrocytes or microglia in the LPBN, at any time point. Thus, our results indicate that while neuropathic pain induces a robust glial reaction in the spinal dorsal horn, it fails to activate glial cells in the LPBN.

Sex-specific differences in alcohol-induced pain sensitization

Pain sensitization is a phenomenon that occurs to protect tissues from damage and recent studies have shown how a variety of non-noxious stimuli included in our everyday lives can lead to pain sensitization. Consumption of large amounts of alcohol over a long period of time invokes alcohol use disorder (AUD), a complex pathological state that has many manifestations, including alcohol peripheral neuropathy (neuropathic pain). We asked if ‘non-pathological’ alcohol consumption can cause pain sensitization in the absence of other pathology? Studies have pointed to glia and other immune cells and their role in pain sensitization that results in cell and sex-specific responses. Using a low-dose and short-term ethanol exposure model, we investigated whether this exposure would sensitize mice to a subthreshold dose of an inflammatory mediator that normally does not induce pain. We observed female mice exhibited specific mechanical and higher thermal sensitivity than males. We also observed an increase in CD68+ macrophages in the ipsilateral dorsal root ganglia (DRG) and Iba1+ microglia in the ipsilateral spinal dorsal horn of animals that were exposed to ethanol and injected with subthreshold inflammatory prostaglandin E2. Our findings suggest that short-term ethanol exposure stimulates peripheral and central, immune and glial activation, respectively to induce pain sensitization. This work begins to reveal a possible mechanism behind the development of alcoholic peripheral neuropathy.

Effect of intrathecal NIS-lncRNA antisense oligonucleotides on neuropathic pain caused by nerve trauma, chemotherapy, or diabetes mellitus

BackgroundBlocking increased expression of nerve injury-specific long non-coding RNA (NIS-lncRNA) in injured dorsal root ganglia (DRG) through DRG microinjection of NIS-lncRNA small hairpin interfering RNA or generation of NIS-lncRNA knockdown mice mitigates neuropathic pain. However, these strategies are impractical in the clinic. This study employed a Food and Drug Administration (FDA)-approved antisense oligonucleotides strategy to examine the effect of NIS-lncRNA ASOs on neuropathic pain. MethodsEffects of intrathecal injection of NIS-lncRNA antisense oligonucleotides on day 7 or 14 after chronic constriction injury (CCI) of the sciatic nerve, fourth lumbar (L4) spinal nerve ligation, or intraperitoneal injection of paclitaxel or streptozotocin on the expression of DRG NIS-lncRNA and C–C chemokine ligand 2 (CCL2, an NIS-lncRNA downstream target) and nociceptive hypersensitivity were examined. We also assessed whether NIS-lncRNA antisense oligonucleotides produced cellular toxicity. ResultsIntrathecal NIS-lncRNA antisense oligonucleotides attenuated CCI-induced mechanical allodynia, heat hyperalgesia, cold hyperalgesia, and ongoing nociceptive responses, without changing basal or acute nociceptive responses and locomotor function. Intrathecal NIS-lncRNA antisense oligonucleotides also blocked CCI-induced increases in NIS-lncRNA and CCL2 in the ipsilateral L3 and L4 DRG and hyperactivities of neurones and astrocytes in the ipsilateral L3 and L4 spinal cord dorsal horn. Similar results were found in antisense oligonucleotides-treated mice after spinal nerve ligation or intraperitoneal injection of paclitaxel or streptozotocin. Normal morphologic structure and no cell loss were observed in the DRG and spinal cord of antisense oligonucleotides-treated mice. ConclusionThese findings further validate the role of NIS-lncRNA in trauma-, chemotherapy-, or diabetes-induced neuropathic pain and demonstrate potential clinical application of NIS-lncRNA antisense oligonucleotides for neuropathic pain management.

Exercise facilitates regeneration after severe nerve transection and further modulates neural plasticity

Patients with severe traumatic peripheral nerve injury (PNI) always suffer from incomplete recovery and poor functional outcome. Physical exercise-based rehabilitation, as a non-invasive interventional strategy, has been widely acknowledged to improve PNI recovery by promoting nerve regeneration and relieving pain. However, effects of exercise on chronic plastic changes following severe traumatic PNIs have been limitedly discussed. In this study, we created a long-gap sciatic nerve transection followed by autograft bridging in rats and tested the therapeutic functions of treadmill running with low intensity and late initiation. We demonstrated that treadmill running effectively facilitated nerve regeneration and prevented muscle atrophy and thus improved sensorimotor functions and walking performance. Furthermore, exercise could reduce inflammation at the injured nerve as well as prevent the overexpression of TRPV1, a pain sensor, in primary afferent sensory neurons. In the central nervous system, we found that PNI induced transcriptive changes at the ipsilateral lumber spinal dorsal horn, and exercise could reverse the differential expression for genes involved in the Notch signaling pathway. In addition, through neural imaging techniques, we found volumetric, microstructural, metabolite, and neuronal activity changes in supraspinal regions of interest (i.e., somatosensory cortex, motor cortex, hippocampus, etc.) after the PNI, some of which could be reversed through treadmill running. In summary, treadmill running with late initiation could promote recovery from long-gap nerve transection, and while it could reverse maladaptive plasticity after the PNI, exercise may also ameliorate comorbidities, such as chronic pain, mental depression, and anxiety in the long term.

FGF 10 Inhibited Spinal Microglial Activation in Neuropathic Pain via PPAR-γ/NF-κB Signaling

Overactivated microglia in the spinal cord leads to neuropathic pain sensitivity. The FGF 10, a Fibroblast Growth Factor (FGFs) that is prevalent in neurons, has been demonstrated to suppress microglial polarization. The objective of this study was to investigate the role of FGF 10 in neuropathic pain and the underlying regulatory mechanisms. Immunofluorescence staining and western blot detection revealed that FGF 10 expression was upregulated in the ipsilateral spinal dorsal horn of Spared Nerve Injury (SNI) rat models and was mainly detected in neurons and microglia. To test the anti-microgliosis actions of FGF 10, SNI rats were intrathecally administered with different concentrations of recombinant FGF 10. Behavioral tests and immunostaining results showed that FGF 10 relieved hyperalgesia in SNI rats and inhibited microglial activity in the ipsilateral spinal dorsal horn in a dose-dependent manner. Besides, BV2 cells were cultured and treated with LPS to activate microglia to explore the underlying mechanisms of FGF 10-induced analgesic effects in vitro. As a result, FGF 10 administration suppressed the LPS-induced microglial augmentation in a dose-dependent manner, followed by increased PPAR-γ and decreased NFκB phosphorylation (p-NFκB) levels. Moreover, PPAR-γ agonist (pioglitazone) and antagonist (GW9662) were administrated into spinal cords of SNI rats, revealing that pioglitazone had similar anti-nociceptive and anti-microglial effects to FGF 10. Conversely, GW9662 reversed all beneficial effects of FGF 10 on SNI rats. In addition, phosphorylated levels of NFκB were reduced by pioglitazone or FGF 10 treatment but raised by GW9662 administration in FGF 10-treated SNI rats. Our findings show that FGF 10 has analgesic effects in rats after peripheral nerve injury and justify the role of PPAR-γ/NFκB signaling in FGF 10-regulated anti-microgliosis.

Upregulation of Hevin contributes to postoperative pain hypersensitivity by inducing neurexin1β/neuroligin1-mediated synaptic targeting of GluA1-containing AMPA receptors in rat dorsal horn

The astrocytes-secreted active molecule, Hevin considerably contributes in the transsynaptic bridge of neurexin1β/neuligin1 in excitatory synapse. Previous studies have demonstrated that activity-dependent synaptic recruitment of spinal neuroligin1 and GluA1-containing AMPA receptors (AMPARs) is involved in incisional, inflammatory and neuropathic pain. Here, we hypothesized that Hevin induced postoperative pain hypersensitivity by enhancing the neurexin1β/neuroligin1-mediated synaptic targeting of GluA1-containing AMPARs in spinal dorsal horns (DH). Our results showed that plantar incision induced significant postoperative pain behavior, which was described by the cumulative pain scores. At 1 d and 3 d post-incision, Hevin expression was considerably elevated in ipsilateral DHs, although it recovered to baseline value at 5 d following the incision. At 1 d post plantar incision, the neurexin1β/neuroligin1 interactions significantly increased in ipsilateral DHs in rats subjected to incision when compared with those in control rats. Intrathecal pretreatments of small interference RNA targeting Hevin substantially suppressed postoperative pain hypersensitivity and reduced the neurexin1β/neurolgin1 interaction as well as the synaptic targeting of GluA1 in ipsilateral spinal DHs. These data suggest that Hevin induced postoperative pain hypersensitivity by enhancing the neurexin1β/neuroligin1 interaction and subsequent synaptic targeting of GluA1-containing AMPARs in ipsilateral spinal DHs. It provides new insights into the role of Hevin-mediated trans-synaptic regulation in postoperative pain hypersensitivity, which would help develop a novel therapeutic strategy.

Antibody Receptor Signaling from Spinal Cord Astrocytes Promotes Neuropathic Pain

Injury to somatosensory nerves can lead to mechanical allodynia, in which normally innocuous touch is perceived as painful. Inflammatory signaling arising from spinal cord glial cells are thought to promote allodynia, but it is unclear what mechanisms control glial activation after injury. Here we demonstrate that Fc gamma receptor IIa (FcγRIIa), an immune receptor for immunoglobulin G (IgG) antibodies, is expressed by astrocytes in the spinal cord and contributes to the development of mechanical allodynia after neuropathic injury. Neuropathic pain in male and female rats was induced by chronic constriction injury (CCI) of the sciatic nerve. Paw withdrawal to innocuous touch was measured by von Frey filaments and paintbrush assay. Intrathecal injections of IgG immune complex (IgG-IC) were used to activate spinal FcγRs. Viral-mediated CRISPR-Cas9 gene editing was used to disrupt Fcgr2a expression in Gfap+ spinal astrocytes in vivo. Gene and protein expression were measured by in situ hybridization, qRT-PCR, and immunohistochemistry. For in vitro experiments, astrocytes were purified by immunopanning and treated with IgG-IC. Activating FcγRs by spinal administration of IgG-IC produces mechanical allodynia. Fcgr2a RNA expression increases specifically in ipsilateral spinal dorsal horn after CCI, and FcγRIIa protein co-localizes primarily with GFAP+ astrocytes. Genetic disruption of Fcgr2a in Gfap+ spinal astrocytes attenuates mechanical allodynia after CCI. Astrocytes respond to IgG-IC by increasing production of pro-inflammatory mediators, including TNF and IL-1β, which are known to influence nociceptor activity. These data suggest FcγRIIa on spinal astrocytes may be activated following peripheral nerve injury and directly contribute to injury-induced tactile pain. These findings expand our understanding of how neuroimmune signaling from glia contribute to the development of mechanical allodynia and suggest that reducing autoantibody IgG signaling at glial FcγRs could be a novel therapeutic target to alleviate suffering from neuropathic pain. Grant support from U.S. Army Medical Research and Materiel Command grants W81XWH-19-1-0160 and W81XWH-16-1-0717 (PMG) Rita Allen Foundation Award in Pain (PMG) University of Texas System Rising STARs Award (PMG) University of Texas MD Anderson Cancer Center start-up funds (PMG) American Australian Association Sir Keith Murdoch Fellowship (MJL).

Hedgehog signaling plays a crucial role in hyperalgesia associated with neuropathic pain in mice.

Neuropathic pain is a debilitating chronic syndrome of the nervous system caused by nerve injury. In Drosophila, the Hedgehog (Hh) signaling pathway is related to increased pain sensitivity (hyperalgesia) but does not affect the baseline nociceptive threshold. In general, the contribution of the Hh signaling pathway to neuropathic pain in vertebrates is a highly debated issue. Alternatively, we investigated the potential role of Hh signaling in mechanical allodynia using a mouse model of neuropathic pain. Seven days after spinal nerve-transection (SNT) surgery, microglial activation increased in the ipsilateral spinal dorsal horn compared with that in the sham group; however, 21 days after surgery, microglial activation decreased. Contrastingly, astrocyte activation in the spinal cord did not differ between the groups. On day 21 of postsurgery, the SNT group showed marked upregulation of sonic hedgehog expression in peripheral glial cells but not in dorsal root ganglion (DRG) neurons. Intrathecal administration of the Hh signaling inhibitor vismodegib attenuated the mechanical allodynia observed on day 21 postsurgery. Conversely, intrathecal treatment with the Hh signaling activator smoothened agonist in naive mice induced mechanical allodynia, which was abolished by the ATP transporter inhibitor clodronate. Moreover, inhibition of Hh signaling by pretreatment with vismodegib significantly reduced ATP secretion and the frequency/number of spontaneous elevations of intracellular calcium ion levels in cultured DRG cells. Thus, the Hh signaling pathway appears to modulate the neural activity of DRG neurons via ATP release, and it plays an important role in sustaining mechanical allodynia and hypersensitivity in a mouse model of neuropathic pain.

Genome-Wide Expression Profiling by RNA-Sequencing in Spinal Cord Dorsal Horn of a Rat Chronic Postsurgical Pain Model to Explore Potential Mechanisms Involved in Chronic Pain.

BackgroundChronic postsurgical pain (CPSP) is common among patients receiving major surgeries. CPSP produces suffering in patients, both physically and mentally. However, the mechanisms underlying CPSP remain elusive. Here, a genome-wide expression profiling of ipsilateral spinal cord dorsal horn (SCDH) was performed to identify potential genes related with CPSP.MethodsA rat skin/muscle incision and retraction (SMIR) model was established to induce CPSP. Immunostaining was used to study glial cell and neuron activation in ipsilateral SCDH of SMIR model rats. RNA sequencing (RNA-Seq), combined with bioinformatics analysis, was undertaken to explore gene expression profiles. qPCR was applied to validate the expression of some representative genes.ResultsThe SMIR model rats developed persistent mechanical allodynia in ipsilateral hindpaw for up to 14 days. Ipsilateral SCDH of SMIR rats showed remarkable glial cell and neuron activation. A number of differentially expressed genes (DEGs) were identified in ipsilateral SCDH of SMIR rats by RNA-Seq. qPCR confirmed expression of some representative DEGs. Bioinformatics indicated that chemical synaptic transmission, sensory perception of pain and neuroactive ligand-receptor interaction were predominant functions. We compared our dataset with human pain-related genes and found that several genes exclusively participate in pain modulation and mechanisms.ConclusionOur study provided novel understandings of the molecular mechanisms possibly contributing to CPSP. These findings may offer new targets for future treatment of CPSP.

High-voltage long-duration pulsed radiofrequency attenuates neuropathic pain in CCI rats by inhibiting Cav2.2 in spinal dorsal horn and dorsal root ganglion

Inclinicalpractice, high-voltage, long-duration pulsed radiofrequency (HL-PRF) is effective for several types of intractable neuropathic pain (NP), but the mechanisms have not been well explored. Cav2.2 channels could increase neuronal excitability and neurotransmission accompanying NP. This study investigated the relationship of the efficacy of HL-PRF on NP with the levels of Cav2.2 in the spinal dorsal horn (SDH) and dorsal root ganglions (DRGs) of chronic constriction injury (CCI) in rats. Sham HL-PRF, GVIA (a specific Cav2.2 channel blocker), HL-PRF, or GVIA + HL-PRF was applied to CCI rats. The results showed: compared with the sham group, the PWT and PWL of CCI rats decreased significantly (P < 0.05), and Cav2.2 expression was elevated significantly in the SDH and DRGs (P < 0.05). Compared with the CCI group, both HL-PRF and ω-conotoxin GVIA treatment reversed the increased PWT and PWL (P < 0.05) and downregulated the overexpression of Cav2.2 in the SDH and DRGs (P < 0.05). Furthermore, PWT, PWL, and the expression of Cav2.2 in the SDH and DRGs were not significantly different among the 3 treatment groups. HL-PRF on L5 DRG reversed the hyperalgesia behavior of NP and reduced the levels of Cav2.2 in the ipsilateral SDH and DRGs in CCI rats. Moreover, the underlying mechanism may be related to the downregulation of CaV2.2 protein levels in both SDH and DRG.